To find optimal conditions for expressing the soluble form of sFv-2F3 and to study the purification and property of its derivative Se-sFv-2F3,the preferred expression conditions were investigated by means of orthogona...To find optimal conditions for expressing the soluble form of sFv-2F3 and to study the purification and property of its derivative Se-sFv-2F3,the preferred expression conditions were investigated by means of orthogonal design. These culture conditions included incubation temperature,inducer concentration, induction time and cell concentration. The evaluation of expression was accomplished by the analysis of whole cell lysates and the yield of soluble sFv-2F3 was calculated according to the analysis of Profinder(FTI-500,Pharmacia). The purification procedure was carried out via a two-step purification procedure consisting of ion-exchange chromatography,followed by immobilized metal affinity chromatography(IMAC). The antioxidant efficacy of Se-sFv-2F3 was demonstrated by the determination of the content of the main product of lipid peroxidation,MDA,the viability of cells and the activity of LDH. We obtained the preferred culture conditions to grow the engineered bacteria and the procedure for preparing soluble sFv-2F3 and confirmed the antioxidant efficacy of Se-sFv-2F3.展开更多
To investigate the treatment effect of 2-selenium bridged β -cyclodextrin(2-SeCD),a GPX mimic,on the stroke of stroke-prone spontaneously hypertensive rats(SHRSP),fifty-two SHRSP of 8-week old were randomly divided i...To investigate the treatment effect of 2-selenium bridged β -cyclodextrin(2-SeCD),a GPX mimic,on the stroke of stroke-prone spontaneously hypertensive rats(SHRSP),fifty-two SHRSP of 8-week old were randomly divided into four groups A,B,C and control group D. The rats of groups A,B,C and D were given 1.0%-1.5% NaCl mass fraction as drinking fluid. After onset of stroke,groups A,B and C were given \{orally\} 16.05,160.5 and 1605 mg·kg -1 ·day -1 of 2-SeCD,respectively,and group D was given water for \{2 weeks.\} The clinical score of stroke,systolic blood pressure(SBP),survival time of rats were recorded and the histopathologic examinations of their brain and carotid artery were made after decapitation. The clinical scores of stroke after treatment with 160.5 mg·kg -1 ·day -1 (Group B) and 1605 mg·kg -1 ·day -1 (Group C) of 2-SeCD are 2.55±0.98 and 1.98±0.79,respectively,those are obviously lower than that of group D(3.41±0.83,p<0.01). The survival days in group B(10.0±8.6) and group C(14.4±7.9) are longer than that for group D(4.7±2.9,p<0.01). The electron microscope study showed that the endothelium of carotid artery was near to normal in group B and group C,while it was seriously injured in control group D and mildly injured in group A. 2-SeCD may effectively be used to treat the stroke for SHRSP.展开更多
The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9...The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions(PCR) and cloned into expression vector pPELB and expressed as a soluble protein in E.coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant(K_A) values range from 1.2×10 4 to 1.7 ×10 5 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.展开更多
文摘To find optimal conditions for expressing the soluble form of sFv-2F3 and to study the purification and property of its derivative Se-sFv-2F3,the preferred expression conditions were investigated by means of orthogonal design. These culture conditions included incubation temperature,inducer concentration, induction time and cell concentration. The evaluation of expression was accomplished by the analysis of whole cell lysates and the yield of soluble sFv-2F3 was calculated according to the analysis of Profinder(FTI-500,Pharmacia). The purification procedure was carried out via a two-step purification procedure consisting of ion-exchange chromatography,followed by immobilized metal affinity chromatography(IMAC). The antioxidant efficacy of Se-sFv-2F3 was demonstrated by the determination of the content of the main product of lipid peroxidation,MDA,the viability of cells and the activity of LDH. We obtained the preferred culture conditions to grow the engineered bacteria and the procedure for preparing soluble sFv-2F3 and confirmed the antioxidant efficacy of Se-sFv-2F3.
文摘To investigate the treatment effect of 2-selenium bridged β -cyclodextrin(2-SeCD),a GPX mimic,on the stroke of stroke-prone spontaneously hypertensive rats(SHRSP),fifty-two SHRSP of 8-week old were randomly divided into four groups A,B,C and control group D. The rats of groups A,B,C and D were given 1.0%-1.5% NaCl mass fraction as drinking fluid. After onset of stroke,groups A,B and C were given \{orally\} 16.05,160.5 and 1605 mg·kg -1 ·day -1 of 2-SeCD,respectively,and group D was given water for \{2 weeks.\} The clinical score of stroke,systolic blood pressure(SBP),survival time of rats were recorded and the histopathologic examinations of their brain and carotid artery were made after decapitation. The clinical scores of stroke after treatment with 160.5 mg·kg -1 ·day -1 (Group B) and 1605 mg·kg -1 ·day -1 (Group C) of 2-SeCD are 2.55±0.98 and 1.98±0.79,respectively,those are obviously lower than that of group D(3.41±0.83,p<0.01). The survival days in group B(10.0±8.6) and group C(14.4±7.9) are longer than that for group D(4.7±2.9,p<0.01). The electron microscope study showed that the endothelium of carotid artery was near to normal in group B and group C,while it was seriously injured in control group D and mildly injured in group A. 2-SeCD may effectively be used to treat the stroke for SHRSP.
文摘The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions(PCR) and cloned into expression vector pPELB and expressed as a soluble protein in E.coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant(K_A) values range from 1.2×10 4 to 1.7 ×10 5 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.
